In the intestine and colon, Hedgehog (Hh) ligands secreted from the epithelium act on multiple target cells of the underlying stroma. Cdl 1b+ as well as Cdl 1c+ myeloid cells express Gli1 in response to Hh ligands and are therefore direct cellular targets of Hh signaling. Evidence from functional mouse models and gene expression studies suggest that intestinal myeloid cells use Hh signals to maintain a tolerogenic phenotype and that altering Hh levels may change myeloid phenotype. This tolerogenic function may hold in humans since a synonymous SNP in the human GLI1 gene that reduces transactivation efficacy is associated with inflammatory bowel disease (IBD) in three large European populations. Aim 1 will define which myeloid subpopulations transduce Hh signals, which genes are the direct targets of Hh signaling in these cells and which enhancers are driving these responses. These genes and enhancers will be compared to all IBD- associated genes to test whether some Hh pathway targets (many of which are inflammatory genes) are also IBD loci. This will have important implications for ongoing GWAS and deep sequencing efforts as our studies will identify myloid genes that can be altered by Hh signaling and will establish a function for some ofthe non-coding DNA associated with these genes. A potential case in point is Nkx2-3, a possible Hh target gene and known IBD-associated gene, which will be examined in Aim 3. Aim 2 will explore whether acute modulation of Hh signal transduction in myeloid cells in vivo (in the presence or absernce of an inflammatory challenge) can alter myeloid phenotypes or change expression of inflammatory genes. Finally, Preliminary, Studies suggest that the upstream region surrounding an IBD-associated Nkx2-3 SNP js rich in Gli binding sites and that Nkx2-3 is induced by Shh and Ihh in isolated intestinal mesenchyme. Thus, Aim 3 will test whether loss of Nkx2-3 affects myeloid inflammatory signaling and/or Hh-inducecl inflammatory responses of myeloid cells. The project coalesces expertise in mouse modeling and Hh signaling (Gumucio), gastric inflammatory responses (Merchant) and gastric and myeloid cell biology (Samuelson, Kao). RELEVANCE (See instructions): Work outlined in this project will examine the role of Hh signal transduction in modulating myeloid phenotype during homeostasis in the intestine and in the context of inflammatory challenge in the intestine and stomach. The work has the potential to impact diagnosis, prognosis and, potentially, treatment of gastrointestinal inflammatory diseases.